Probing Local Force Propagation in Tensed Fibrous Gels.

Fibrous hydrogels are a key component of soft animal tissues. They support cellular functions and facilitate efficient mechanical communication between cells. Due to their nonlinear mechanical properties, fibrous materials display non-trivial force propagation at the microscale, that is enhanced compared to that of linear-elastic materials. In the body, tissues are constantly subjected to external loads that tense or compress them, modifying their micro-mechanical properties into an anisotropic state. However, it is unknown how force propagation is modified by this isotropic-to-anisotropic transition. Here, force propagation in tensed fibrin hydrogels is directly measured. Local perturbations are induced by oscillating microspheres using optical tweezers. 1-point and 2-point microrheology are combined to simultaneously measure the shear modulus and force propagation. A mathematical framework to quantify anisotropic force propagation trends is suggested. Results show that force propagation becomes anisotropic in tensed gels, with, surprisingly, stronger response to perturbations perpendicular to the axis of tension. Importantly, external tension can also increase the range of force transmission. Possible implications and future directions for research are discussed. These results suggest a mechanism for favored directions of mechanical communication between cells in a tissue under external loads.

Transmembrane proteins tetraspanin 4 and CD9 sense membrane curvature.

Multiple membrane-shaping and remodeling processes are associated with tetraspanin proteins by yet unknown mechanisms. Tetraspanins constitute a family of proteins with four transmembrane domains present in every cell type. Prominent examples are tetraspanin4 and CD9, which are required for the fundamental cellular processes of migrasome formation and fertilization, respectively. These proteins are enriched in curved membrane structures, such as cellular retraction fibers and oocyte microvilli. The factors driving this enrichment are, however, unknown. Here, we revealed that tetraspanin4 and CD9 are curvature sensors with a preference for positive membrane curvature. To this end, we used a biomimetic system emulating membranes of cell retraction fibers and oocyte microvilli by membrane tubes pulled out of giant plasma membrane vesicles with controllable membrane tension and curvature. We developed a simple thermodynamic model for the partitioning of curvature sensors between flat and tubular membranes, which allowed us to estimate the individual intrinsic curvatures of the two proteins. Overall, our findings illuminate the process of migrasome formation and oocyte microvilli shaping and provide insight into the role of tetraspanin proteins in membrane remodeling processes.

Correction: Live cell single molecule tracking and localization microscopy of bioorthogonally labeled plasma membrane proteins.

Correction for 'Live cell single molecule tracking and localization microscopy of bioorthogonally labeled plasma membrane proteins' by Andres I. König et al., Nanoscale, 2020, 12, 3236-3248, DOI: 10.1039/C9NR08594G.

Live cell single molecule tracking and localization microscopy of bioorthogonally labeled plasma membrane proteins.

Tracking the localization and mobility of individual proteins in live cells is key for understanding how they mediate their function. Such information can be obtained from single molecule imaging techniques including as Single Particle Tracking (SPT) and Single Molecule Localization Microscopy (SMLM). Genetic code expansion (GCE) combined with bioorthogonal chemistry offers an elegant approach for direct labeling of proteins with fluorescent dyes, holding great potential for improving protein labeling in single molecule applications. Here we calibrated conditions for performing SPT and live-SMLM of bioorthogonally labeled plasma membrane proteins in live mammalian cells. Using SPT, the diffusion of bioorthogonally labeled EGF receptor and the prototypical Shaker voltage-activated potassium channel (Kv) was measured and characterized. Applying live-SMLM to bioorthogonally labeled Shaker Kv channels enabled visualizing the plasma membrane distribution of the channel over time with ∼30 nm accuracy. Finally, by competitive labeling with two Fl-dyes, SPT and live-SMLM were performed in a single cell and both the density and dynamics of the EGF receptor were measured at single molecule resolution in subregions of the cell. We conclude that GCE and bioorthogonal chemistry is a highly suitable, flexible approach for protein labeling in quantitative single molecule applications that outperforms current protein live-cell labeling approaches.

Synaptotagmin-1 and Doc2b Exhibit Distinct Membrane-Remodeling Mechanisms.

Synaptotagmin-1 (Syt1) is a calcium sensor protein that is critical for neurotransmission and is therefore extensively studied. Here, we use pairs of optically trapped beads coated with SNARE-free synthetic membranes to investigate Syt1-induced membrane remodeling. This activity is compared with that of Doc2b, which contains a conserved C2AB domain and induces membrane tethering and hemifusion in this cell-free model. We find that the soluble C2AB domain of Syt1 strongly affects the probability and strength of membrane-membrane interactions in a strictly Ca2+- and protein-dependent manner. Single-membrane loading of Syt1 yielded the highest probability and force of membrane interactions, whereas in contrast, Doc2b was more effective after loading both membranes. A lipid-mixing assay with confocal imaging reveals that both Syt1 and Doc2b are able to induce hemifusion; however, significantly higher Syt1 concentrations are required. Consistently, both C2AB fragments cause a reduction in the membrane-bending modulus, as measured by a method based on atomic force microscopy. This lowering of the energy required for membrane deformation may contribute to Ca2+-induced fusion.

Probing cellular mechanics with acoustic force spectroscopy.

A large number of studies demonstrate that cell mechanics and pathology are intimately linked. In particular, deformability of red blood cells (RBCs) is key to their function and is dramatically altered in the time course of diseases such as anemia and malaria. Due to the physiological importance of cell mechanics, many methods for cell mechanical probing have been developed. While single-cell methods provide very valuable information, they are often technically challenging and lack the high data throughput needed to distinguish differences in heterogeneous populations, while fluid-flow high-throughput methods miss the accuracy to detect subtle differences. Here we present a new method for multiplexed single-cell mechanical probing using acoustic force spectroscopy (AFS). We demonstrate that mechanical differences induced by chemical treatments of known effect can be measured and quantified. Furthermore, we explore the effect of extracellular vesicles (EVs) uptake on RBC mechanics and demonstrate that EVs uptake increases RBC deformability. Our findings demonstrate the ability of AFS to manipulate cells with high stability and precision and pave the way to further new insights into cellular mechanics and mechanobiology in health and disease, as well as potential biomedical applications.

The effect of the serum corona on interactions between a single nano-object and a living cell.

Nanoparticles (NPs) which enter physiological fluids are rapidly coated by proteins, forming a so-called corona which may strongly modify their interaction with tissues and cells relative to the bare NPs. In this work the interactions between a living cell and a nano-object, and in particular the effect on this of the adsorption of serum proteins, are directly examined by measuring the forces arising as an Atomic Force Microscope tip (diameter 20 nm) - simulating a nano-object - approaches and contacts a cell. We find that the presence of a serum protein corona on the tip strongly modifies the interaction as indicated by pronounced increase in the indentation, hysteresis and work of adhesion compared to a bare tip. Classically one expects an AFM tip interacting with a cell surface to be repelled due to cell elastic distortion, offset by tip-cell adhesion, and indeed such a model fits the bare-tip/cell interaction, in agreement with earlier work. However, the force plots obtained with serum-modified tips are very different, indicating that the cell is much more compliant to the approaching tip. The insights obtained in this work may promote better design of NPs for drug delivery and other nano-medical applications.